Because the sensitivity of our "nested" PCR method is at least equivalent to the chimpanzee model, we proceeded to use the method to assay HCV RNA in plasma derivatives such as IGIV, IG, and AHF. An ultracentrifugal method (50,000 rpm for 3 h with a 70 Ti rotor, ca. 180,000 x g) was developed to concentrate "virus" in the pellet while at least 90% of the protein remained in the supernatant. We assayed seven experimental lots, produced by 7 manufacturers from Source Plasma (2,887 units) that had been screened for anti-HCV (anti-c100-3). HCV RNA was detected in one of these experimental lots, even through there was no evidence of hepatitis C transmission when this lot, along with the other six PCR-negative lots, was infused into 3 chimpanzees. We also analyzed 32 routine lots of IGIV (prepared from plasma not screened for anti-HCV) from the 7 manufacturers. HCV RNA was detected in four of these lots, all from one manufacturer (the same manufacturer whose experimental IGIV lot, produced from screened plasma, was positive). These five positive lots were determined by limiting dilution analysis to have from 10 to 250 PCR U/g IgG. It is of interest that 4 IGIV lots produced by this manufacturer from recovered plasma was negative for HCV RNA, whereas 4 out of 5 lots derived from Source Plasma were positive. One lot of VZIG prepared from recovered plasma by the same manufacturer was also negative. This may be due to the lower anti-HCV reactive rate, hence, the lower viral load, in recovered plasma. Two intramuscular IG lots and two older AHF lots were assayed and found to contain 30 and 260 PCR U/g IgG, and 0.1 and 4 PCR U/IU FVIII, respectively. One lot of Coagulation Factor IX (Human) (purified, heated in heptane) was assayed and tested negative. Preliminary data obtained from studies utilizing Rnase A digestion suggest that the HCV RNA presented in immune globulins may be as a "naked" RNA rather than intact virus; this may explain how viral RNA can be present in some immune globulin products without transmitting hepatitis C to recipients. Detection and quantitation of HCV RNA and its digestibility by Rnase A in plasma fractionation products will be continued.